Molecular Detection of Phytophthora spp. P. ramorum, P. nemorosa, and P. pseudosyringae   

The described PCR detection system is based on amplification of the spacer region between the mitochondrially-encoded cox I and II genes of Phytophthora using genus-specific primers developed from conserved regions of the flanking genes.  A two-step multiplex amplification procedure is used for determining if a Phytophthora sp. was present in symptomatic plant tissue and clarifying if it was P. ramorum or P. nemorosa or P. pseudosyringae, two additional species commonly encountered in samples from forest ecosystems in California.  The first round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for PCR amplification and a second primer pair specific for amplification of sequences from Phytophthora spp.   The plant primers amplified the desired amplicon size in all the plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair.  Using DNA from purified cultures the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species.  The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species.  With the exception of one plant species the resulting amplicons were smaller than the Phytophthora genus-specific amplicon.  The products of the first round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa or P. pseudosyringae.  These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum this included 24 isolates from California, Germany and the Netherlands.  Using purified pathogen DNA the limit of detection for P. ramorum using this marker system was approximately 2.0 fg of total DNA.  However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure the sensitivity of detection was reduced by 100 to 1000 fold depending on the plant species.  Using a dilution series of purified DNA from P. ramorum the nested mitochondrial marker system was found to have a  level of sensitivity comparable to the ITS marker system. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions, and for P. ramorum, amplification with a previously published rDNA ITS species-specific primer pair.   Results were compared and validated with three different brands of thermalcyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions.  The specificity of the Phytophthora genus-specific primers suggests they will have utility for pathogen detection in other Phytophthora pathosystems and the variability encountered in the spacer regions should be useful for constructing additional species-specific primers.

·   References

Martin, F.N, Tooley, P.W. and Blomquist, C. (2004) Molecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two additional species commonly recovered from diseased plant material.  Phytopathology 94: 621-631 (.pdf reprint)

Tooley, P.W., Martin, F.N., Carras, M.M. and Frederick, R.D. (2006) Real-time fluorescent PCR detection of Phytophthora ramorum and Phytophthora pseudosyringae using mitochondrial gene regions.  Phytopathology 96: 336-345 (pdf reprint)

P.  ramorum conventional PCR amplification procedure

  • Modifications to amplification procedure
  • Potential alternative plant primers 
  • Detection of P. ramorum in a single amplification
  • Real-time detection of P. ramorum

P.ramorum TaqMan real-time PCR detection procedure

Additional details

§         Plant primers

§         Phytophthora genus-specific primer pair  

§         P.ramorum species-specific primer pair

·   Multiplex amplification

·   Limit of detection

·   Sensitivity compared to the ITS marker system 

·   Amplification from symptomatic plant tissue

§         Additional species-specific primers nested in the genus-specific amplicon

  • P. nemorosa species-specific primer pair
  • P. pseudosyringae species-specific primer pair
  • Additional species-specific primers
  • Sequence alignments of the Phytophthora genus-specific amplicons